ArticleAmino acids2016
Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.
Article in Amino acids, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed, 60 citations in OpenAlex.
- Development and Integrated Application of the Multi-Attribute Method (MAM) in Quality Control of Biotechnological Drugs.Biotechnology and bioengineering · 2026Review
- On-column thiol protection strategy to control charge and stability of ThiomAb.Antibody therapeutics · 2026Article
- Strategies for in-depth and simultaneous analysis of N- and O-glycoproteome of platelets uncovering sex-specific signatures of human platelets.Analytical and bioanalytical chemistry · 2026Article
- Epitope and Paratope Mapping of a SUMO-Remnant Antibody Using Cross-Linking Mass Spectrometry and Molecular Docking.Journal of proteome research · 2025Article
- Revisiting the Effect of Trypsin Digestion Buffers on Artificial Deamidation.Journal of the American Society for Mass Spectrometry · 2025Article
- Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry.ACS measurement science au · 2024Review
- Site-Specific Conjugation of Native Antibody: Transglutaminase-Mediated Modification of a Conserved Glutamine While Maintaining the Primary Sequence and Core Fc Glycan via Trimming with an Endoglycosidase.Bioconjugate chemistry · 2024Article
- Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins.Journal of proteome research · 2023Article
- Article
- An Improved Isotope Labelling Method for Quantifying Deamidated Cobratide Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry.Molecules (Basel, Switzerland) · 2022Article
- Understanding the pathway and kinetics of aspartic acid isomerization in peptide mapping methods for monoclonal antibodies.Analytical and bioanalytical chemistry · 2021Article
- ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping.Molecular & cellular proteomics : MCP · 2020Article
- Site-specific conjugation of native antibody.Antibody therapeutics · 2020Article
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- Comparison of ELISA and HPLC-MS methods for the determination of exenatide in biological and biotechnology-based formulation matrices.Journal of pharmaceutical analysis · 2019Review
- Sequence and Solution Effects on the Prevalence of d-Isomers Produced by Deamidation.ACS chemical biology · 2017Article
- LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics.Journal of visualized experiments : JoVE · 2017Article
- Microwave-assisted cross-linking of milk proteins induced by microbial transglutaminase.Scientific reports · 2016Article
- Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics.Journal of the American Society for Mass Spectrometry · 2016Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH's (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.